Bottom Line:
A total of 49 spots were analysed revealing 25 proteins differentially expressed, among them many proteins involved in calcium regulation.Remarkably, a group of proteins dealing with calcium metabolism and calcium regulation has been found to be lost through peritoneal dialysate effluent, giving thus a potential explanation to the calcification of soft tissues in patients subjected to peritoneal dialysis and kidney injury.Comparison of literature dealing with PD is difficult due to differences in sample treatment and analytical methodologies.

Background: Peritoneal dialysis (PD) is a form of renal replacement used for advanced chronic kidney disease. PD effluent holds a great potential for biomarker discovery for diagnosis and prognosis. In this study a novel approach to unravelling the proteome of PD effluent based-on dithiothreitol depletion followed by 2D-SDS-PAGE and protein identification using tandem mass spectrometry is proposed.

Results: A total of 49 spots were analysed revealing 25 proteins differentially expressed, among them many proteins involved in calcium regulation.

Conclusions: Remarkably, a group of proteins dealing with calcium metabolism and calcium regulation has been found to be lost through peritoneal dialysate effluent, giving thus a potential explanation to the calcification of soft tissues in patients subjected to peritoneal dialysis and kidney injury. Comparison of literature dealing with PD is difficult due to differences in sample treatment and analytical methodologies.

Mentions:
In order to analyze the differences among the patients, 2D gels were carried out by triplicate for each patient. A total of 100 μg of protein was loaded onto pH 3–10 strips, and then proteins were visualized with CBB. A representative gel is presented in Figure 1. The 2D gels, were analysed using Progenesis SameSpots software as described in Section 2.9, and 49 spots were detected and analysed (Figure 1B). Gel spots were excised and subjected to in-gel digestion and MALDI-TOF/TOF analysis. Detailed information on the identified proteins is given in Additional file2: Table S1SM and Additional file3: Table S2SM. A total of 49 spots were excised and analysed, rendering 49 identifications, after removing redundancy only 25 different proteins were identified, see Figure 2. Some proteins, such as Ig kappa chain C region as well as Fibrinogen beta chain were found in as much as 7 and 9 spots, respectively. This redundancy can be linked with the existence of post- translational modifications (PTMs) or the presence of proteases in the PDE, which might be responsible for the proteolysis of the proteins, rendering this way a solution rich in protein fragments. These two effects would lead to identify the same protein in different spots. Therefore, and for future works, it is recommended to collect the PDE with a cocktail of protease inhibitors as well as the analysis of PTMs.

Mentions:
In order to analyze the differences among the patients, 2D gels were carried out by triplicate for each patient. A total of 100 μg of protein was loaded onto pH 3–10 strips, and then proteins were visualized with CBB. A representative gel is presented in Figure 1. The 2D gels, were analysed using Progenesis SameSpots software as described in Section 2.9, and 49 spots were detected and analysed (Figure 1B). Gel spots were excised and subjected to in-gel digestion and MALDI-TOF/TOF analysis. Detailed information on the identified proteins is given in Additional file2: Table S1SM and Additional file3: Table S2SM. A total of 49 spots were excised and analysed, rendering 49 identifications, after removing redundancy only 25 different proteins were identified, see Figure 2. Some proteins, such as Ig kappa chain C region as well as Fibrinogen beta chain were found in as much as 7 and 9 spots, respectively. This redundancy can be linked with the existence of post- translational modifications (PTMs) or the presence of proteases in the PDE, which might be responsible for the proteolysis of the proteins, rendering this way a solution rich in protein fragments. These two effects would lead to identify the same protein in different spots. Therefore, and for future works, it is recommended to collect the PDE with a cocktail of protease inhibitors as well as the analysis of PTMs.

Bottom Line:
A total of 49 spots were analysed revealing 25 proteins differentially expressed, among them many proteins involved in calcium regulation.Remarkably, a group of proteins dealing with calcium metabolism and calcium regulation has been found to be lost through peritoneal dialysate effluent, giving thus a potential explanation to the calcification of soft tissues in patients subjected to peritoneal dialysis and kidney injury.Comparison of literature dealing with PD is difficult due to differences in sample treatment and analytical methodologies.

Background: Peritoneal dialysis (PD) is a form of renal replacement used for advanced chronic kidney disease. PD effluent holds a great potential for biomarker discovery for diagnosis and prognosis. In this study a novel approach to unravelling the proteome of PD effluent based-on dithiothreitol depletion followed by 2D-SDS-PAGE and protein identification using tandem mass spectrometry is proposed.

Results: A total of 49 spots were analysed revealing 25 proteins differentially expressed, among them many proteins involved in calcium regulation.

Conclusions: Remarkably, a group of proteins dealing with calcium metabolism and calcium regulation has been found to be lost through peritoneal dialysate effluent, giving thus a potential explanation to the calcification of soft tissues in patients subjected to peritoneal dialysis and kidney injury. Comparison of literature dealing with PD is difficult due to differences in sample treatment and analytical methodologies.